The cornea forms the anterior surface of the eye and is responsible for most of the eye’s refractive power. Injury to the outermost layer of the cornea, a non-keratinized stratified squamous epithelium, triggers a transient rise in intracellular calcium concentration that propagates radially from the wound. This calcium mobilization is initiated by the binding of nucleotides such as adenosine triphosphate (ATP), which are released from cells ruptured by the injury, to purinergic receptors (purinoreceptors) on undamaged cells near the wound. Downstream effects of this injury-induced "calcium wave" are generally thought to include the activation of signaling pathways that promote wound healing. However, the specific contributions of individual purinergic receptors to the overall wound response have in most cases not been well characterized. Purinoreceptors are classified into two broad categories: the P2Y class of G protein-coupled receptors, which act through second messengers to release calcium into the cytosol from the endoplasmic reticulum, and the P2X class of ligand-gated ionotropic receptors, which release calcium into the cytosol from the extracellular environment. Previously, our lab established the importance of the P2Y2 receptor to corneal epithelial wound healing by showing that P2Y2 activation makes a substantial contribution to the overall wound-induced calcium response, particularly in cells back from the leading edge, and promotes cell migration after injury. P2Y2 activation was also found to promote the phosphorylation of proteins involved in focal adhesions, which are multi-protein complexes that facilitate cell migration by transmitting the forces generated by the actin cytoskeleton to the extracellular environment. More recently, our lab has begun to demonstrate that P2X7 may play an equally important, yet distinct and perhaps complementary role in corneal epithelial wound healing. For instance, P2X7 was found to strongly influence the intensity of the injury-induced calcium response in cells immediately adjacent to the wound, and treatment with the P2X7 inhibitor oxidized ATP (oxATP) was shown to impair migration after injury both in vitro and in ex vivo rat corneas. Additionally, immunofluorescence of cells fixed eight hours after injury revealed an altered actin cytoskeletal architecture and localization of the focal adhesion proteins talin and vinculin in oxATP-treated cells compared to control cells. The goal of the present study was to further characterize P2X7’s role in the overall response to injury by using live cell imaging to examine actin cytoskeletal rearrangements and focal adhesion dynamics after injury under both control conditions and conditions of P2X7 inhibition. Human corneal limbal epithelial (HCLE) cells were transduced to express either actin or talin tagged with green fluorescent protein (GFP), grown into confluent monolayers, and scratch wounded in the presence or absence of oxATP. Cells at the leading edge of the wound were imaged using confocal microscopy every 10 minutes for 4 hours beginning 0.5 hours after injury. Analysis of the resulting actin-GFP movies revealed trends toward delayed extension of filopodia in oxATP-treated cells relative to control cells, as well as complex changes in the number of filopodia per cell over time. Additionally, while both groups formed lamella containing thick actin bundles that were oriented perpendicularly to the direction of migration, in oxATP-treated cells the formation of these structures was delayed. Furthermore, in oxATP-treated cells these actin bundles tended to persist once formed. This was in contrast to control cells, in which they tended to turn over to be replaced by thinner and shorter actin bundles that were oriented more obliquely relative to the direction of migration. Finally, analysis of talin-GFP movies demonstrated that focal adhesion lifespan was extended in oxATP-treated cells compared to control cells. Focal adhesions in oxATP-treated cells also exhibited a greater propensity to merge together or split apart, further suggesting impaired focal adhesion turnover. Overall, these findings suggest that P2X7 plays a critical role in promoting migration after corneal epithelial injury by coordinating rapid rearrangements of the actin cytoskeleton and turnover of focal adhesions at the leading edge.

Cells are dynamic systems that generate and respond to forces through the complex interplay between biochemical and mechanical regulations. Since cellular processes often happen at the molecular level and are challenging to be observed under in vivo conditions due to limitations in optical microscopy, multiple analysis tools have been developed to gain insight into those processes. One of the ways to characterize these mechanical properties is by measuring their persistence length, the average length over which filaments stay straight. There are several approaches in the literature for measuring the persistence length of the filaments, including Fourier analysis of images obtained using fluorescence microscopy. Here, we show how curvature can be used to quantify local deformations of cell shape and cellular components. We develop a novel technique, called curvature analysis, to measure the stiffness of bio-filaments from fluorescent images. We test our predictions with Monte-Carlo generated filaments. We also apply our approach to microtubules and actin filaments obtained from in vitro gliding assay experiments with high densities of non-functional motors. The presented curvature analysis is significantly more accurate compared to existing approaches for small data sets. To study the effect of motors on filament deformations and velocities observed in gliding assays with functional and non-functional motors, we developed Langevin dynamics simulations of on glass and lipid surfaces. We found that generally the gliding velocity increases with an increase in motor density and a decrease in diffusion coefficient, and that motor density and diffusion coefficient have no clear effect on filament curvatures, except at a very low diffusion coefficients. Finally, we provide an ImageJ plugin to make curvature and persistence length measurements more accessible to everyone.

Les nanotubes de tunnellisation (TNT) sont des structures riches en F-actine qui relient des cellules distantes, permettant le transport de nombreux composants cellulaires (des vésicules, des organites etc.). Les TNT sont impliqués dans des processus cellulaires clés, tels que le développement, l'immunité et la régénération des tissus, mais également dans la transmission de divers agents pathogènes. Plusieurs facteurs moléculaires ont été identifiés pour participer à la formation de la formation de TNT. Le complexe de l'exocyste est l'un des premiers facteurs moléculaires impliqués dans cette formation. Il est impliqué dans l’attachement des vésicules sécrétoires, suggère que les protéines qui régulent le trafic vésiculaire pourraient jouer un rôle dans la formation de TNT. Nous avons émis l'hypothèse que la formation de TNT est modulée par des protéines qui participent à la fois à la régulation du trafic vésiculaire et au remodelage du cytosquelette d'actine, deux processus qui sont essentiels pour la formation de ces structures. Comme les Rab-GTPase sont les principaux régulateurs du trafic vésiculaire et participent à la régulation du cytosquelette d'actine, nous avons examiné le rôle de cette famille de protéines dans la formation de TNT. Tout d'abord, nous avons effectué un criblage de plusieurs protéines de Rab pour son effet sur le transfert de vésicule dépendant de TNT. Nous avons constaté que Rab8a, Rab11a et Rab35 ont un effet positif sur le transfert de vésicule. Surexpression de Rab8a et Rab11a augmentait également le nombre de cellules connectées au TNT. Lors de la surexpression de VAMP3 (une autre protéine impliquée dans le trafic vésiculaire), augmentait du nombre de cellules connectées au TNT. Une analyse plus poussée a montré que les trois protéines (Rab11a, Rab8a et VAMP3), ont un effet sur la formation de TNT de manière cascade. Pour établir une relation entre Rab11a et Rab8a, nous avons vérifié le rôle de Rabin8 sur la formation de TNT (une protéine qui interagit avec Rab11 et qui active Rab8) et nous n’avons observé aucun effet dans la formation de TNT. De plus, nous avons vérifié une autre protéine dont la fonction est similaire à Rabin8, à savoir GRAB (facteur d’échange de nucléotide de guanine-GAP- pour Rab3A) et son rôle dans la formation de TNT. Surexpression de GRAB augmente la formation de TNT, mais qu’elle agit de manière indépendante de Rab11 et Rab8a pour réguler la formation de TNT. L'analyse de Rab35-GTP, impliquée dans le recyclage des endocytes, la cytokinèse et la croissance des neurites, augmente la formation de TNT. La croissance des neurites est nécessaire pour la connectivité neuronale et le recyclage des vésicules joue un rôle crucial dans ce processus. Rab35 interagit avec plusieurs protéines impliquées dans le trafic vésiculaire, telles que ACAP2 (GAP de ARF6), MICAL-L1 (molécule interagissant avec CasL-like1 et participe dans le recyclage des vésicules) EHD1 (un ciseau moléculaire) qui participe dans la scission de la vésicule). Sur les endosomes positifs pour ARF6, Rab35 recrute ACAP2 et MICAL-L1 et forme un complexe qui se lie à EHD1 pour réguler la croissance des neurites. Nos données suggèrent fortement que ces effecteurs pourraient également être impliqués dans la formation de TNT. Individuellement, ACAP2, EHD1 et ARF6-GDP régulent la formation de TNT de manière positive et MICAL-L1 ne montre aucun effet sur les TNT. En outre, des données préliminaires indiquent que Rab35 et EHD1 agissent dans un mécanisme en cascade pour réguler la formation de TNT, suggérant que la formation de TNT et la croissance des neurites peuvent agir de manière similaire. Les molécules identifiées constituent des cibles moléculaires potentielles pour les thérapies visant à bloquer la propagation d'agents pathogènes transférés à travers les TNT. Cette étude prouve que les protéines jouant un rôle dans le trafic vésiculaire et la croissance des neurites participent également à la formation de TNT. / Tunneling nanotubes (TNTs) are F-actin rich structures that connect distant cells, allowing the transport of many cellular components, including vesicles, organelles and different kind of molecules. TNTs are implicated in key cellular processes, such as development, immunity and tissue regeneration, but also in the transmission of various pathogens. Several molecular factors have been identified to participate in the regulation of TNT formation. One of the early molecular factors that is implicated in TNT formation is the exocyst complex. This complex is also involved in the tethering of secretory vesicles during secretion, which suggest that proteins that regulate vesicle trafficking could have a role in TNT formation. We have hypothesized that the formation of TNTs is modulated by proteins that participate in both, the regulation of vesicle trafficking and the remodelling of the actin cytoskeleton, and that these two processes are key for the formation of these structures. Since Rab GTPases are the major regulators of vesicle trafficking and also participate in actin cytoskeleton regulation, we examined the role of this protein family in TNT formation. First, we performed a screening of several different Rab proteins for its effect on TNT-dependent vesicle transfer. We found that Rab8a, Rab11a and Rab35 have a positive effect on vesicle transfer. Additional studies demonstrated that Rab8a and Rab11a overexpression also increase the number of TNT connected cells. Upon overexpression of VAMP3 (another protein involved in vesicle trafficking), we also observed an increase in the number of TNT connected cells. Further analysis showed that all three proteins, i.e. Rab11a, Rab8a and VAMP3, show an effect on TNT formation in a cascade dependent manner. To establish a relationship between Rab11a and Rab8a, we checked the role of Rabin8 (a protein that interacts with Rab11 and activates Rab8), on TNT formation and we found that it has no role in TNT formation. Additionally, we checked another protein whose function is similar to Rabin8, i.e. GRAB (guanine nucleotide exchange factor for Rab3A) and its role in TNT formation. The results show that GRAB overexpression increases TNT formation, but it acts in a pathway independent of Rab11 and Rab8a to regulate TNT formation. The analysis of Rab35, a protein involved in endocytic recycling, cytokinesis and neurite outgrowth, showed that the GTP-Rab35 bound form also increases TNT formation. Neurite outgrowth is an essential process in order to establish neural connectivity and vesicle recycling plays a crucial role in this process. Rab35 interacts with several proteins, that are involved in vesicle trafficking such as such as ACAP2 (acts as GAP of ARF6), MICAL-L1 (molecule interacting with CasL-like 1, which plays a role in vesicle recycling) EHD1 (a molecular scissor that has a role in vesicle scission). At the ARF6 positive endosomes, Rab35 recruits ACAP2 and MICAL-L1, and forms a complex that binds to EHD1 to regulate neurite outgrowth.Our data strongly suggest that these effectors may also be involved in the formation of TNTs. Individually, ACAP2, EHD1 and ARF6-GDP regulate TNT formation in a positive manner. But MICAL-L1 overexpression in cells shows no effect on TNTs. Also, preliminary data, indicates that Rab35 and EHD1 acts in a cascade mechanism to regulate TNT formation. This indicates that TNT formation and neurite outgrowth may act in a similar, but not exact pathway. The molecules identified here that have a role in TNT formation, constitute potential molecular targets for therapies aiming to block the spreading of pathogens that transfer through TNTs.This study proves that proteins that have a role in vesicle trafficking and neurite outgrowth, such as Rab proteins, are also involved in TNT formation.

Indiana University-Purdue University Indianapolis (IUPUI) / Insulin-stimulated glucose uptake into skeletal muscle cells requires translocation of the glucose transporter-4 (GLUT4) from the cell interior to the plasma membrane. Insulin-stimulated GLUT4 vesicle translocation is dysregulated in Type 2 diabetes (T2D). The Group I p21–activated kinase (PAK1) is a required element in insulin-stimulated GLUT4 vesicle translocation in mouse skeletal muscle in vivo, although its placement and function(s) in the canonical insulin signaling cascade in skeletal muscle cells, remain undetermined. Therefore, the objective of my project is to determine the molecular mechanism(s) underlying the requirement for PAK1 in the process of insulin-stimulated GLUT4 vesicle translocation and subsequent glucose uptake by skeletal muscle cells. Toward this, my studies demonstrate that the pharmacological inhibition of PAK1 activation blunts insulin-stimulated GLUT4 translocation and subsequent glucose uptake into L6-GLUT4myc skeletal myotubes. Inhibition of PAK1 activation also ablates insulin-stimulated F-actin cytoskeletal remodeling, a process known to be required for mobilizing GLUT4 vesicles to the plasma membrane. Consistent with this mechanism, PAK1 activation was also required for the activation of cofilin, another protein implicated in F-actin remodeling. Interestingly, my studies reveal a novel molecular mechanism involving PAK1 signaling to p41-ARC, a regulatory subunit of the cytoskeletal Arp2/3 complex, and its interactions with another cytoskeletal factor, N-WASP, to elicit the insulin-stimulated F-actin remodeling in skeletal muscle cells. Pharmacological inactivation of N-WASP fully abrogated insulin-stimulated GLUT4 vesicle translocation to the cell surface, coordinate with blunted F-actin remodeling. Furthermore, my studies revealed new insulin-induced interactions amongst N WASP, actin, p41-ARC and PAK1; inactivation of PAK1 signaling blocked these dynamic interactions. Taken together, the above studies demonstrate the significance of PAK1 and its downstream signaling to F-actin remodeling in insulin-stimulated GLUT4 vesicle translocation and glucose uptake, revealing new signaling elements that may prove to be promising targets for future therapeutic design.

Indiana University-Purdue University Indianapolis (IUPUI) / Age-related hearing loss is an acute health problem affecting people worldwide, often arising due to defects in the proper functioning of sensory hair cells in the inner ear. The apical surface of sensory hair cells contains actin-based protrusions known as stereocilia, which detect sound and head movements. Since hair cells are not regenerated in mammals, it is important to maintain the functioning of stereocilia for the life of an organism to maintain hearing ability. The actin filaments within a stereocilium are extensively crosslinked by various actin crosslinking proteins, which are important for stereocilia development and maintenance. Multiple studies have shown that the stereocilia actin core is exceptionally stable whereas actin is dynamic only at the tips of stereocilia. However, whether the actin crosslinking proteins, which are nearly as abundant as actin itself, are similarly stable or can freely move in and out of the core remains unknown. Loss or mutation of crosslinkers like plastin-1, fascin-2, and XIRP2 causes progressive hearing loss along with stereocilia degeneration while loss of espin prevents stereocilia from even developing properly. Do these phenotypes stem from an unstable stereocilia core? Does crosslinking confer stability to the core? To address these questions, we generated novel transgenic reporter lines to monitor the dynamics of actin in mice carrying fascin-2R109H mutation and espin null mice and also to study the dynamics of actin crosslinkers, in vivo and ex-vivo. We established that actin crosslinkers readily exchange within the highly stable F-actin structure of the stereocilia core. In addition, we determined that stereocilia degeneration in mice carrying fascin-2R109H mutation and espin null mice could possibly occur due to a less stable actin core. These studies suggest that dynamic crosslinks stabilize the core to maintain proper stereocilia functioning. Future work warrants understanding the reason behind the importance of dynamic crosslinks within a stable stereocilia core. Actin stability not only depends on actin crosslinkers, but also on actin filament composition as evident from distinct stereocilia degeneration and progressive hearing loss patterns in hair-cell specific knockout of actin isoforms. Although beta- and gamma- actin polypeptide sequences differ by only 14 four amino acids, whether the latter determine the unique function of each cytoplasmic actin isoform was previously unknown. Here we determined that these four critical amino acids determine the unique functional importance of beta-actin isoform in sensory hair cells. Taken together, our study demonstrates that actin cytoskeletal proteins are important for the morphogenesis and maintenance of stereocilia.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18657号 / 工博第3966号 / 新制||工||1610(附属図書館) / 31571 / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 岩田 博夫, 教授 木村 俊作, 教授 秋吉 一成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

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Elevated eosinophil counts in the circulation and/or tissues is considered a clinical feature and biomarker of several chronic airway diseases including asthma. As such, many therapeutic biologics for asthma developed within the past decade target eosinophil recruitment to and accumulation in the airways to mixed success. Although the nature of adhesive interactions and directional migration of eosinophils has been well studied, there remains a lack of comprehensive understanding regarding the components which modulate eosinophil movement from the blood into respiratory tissues that impacts the efficacy of these clinical studies; therefore, continued research in this area may reveal novel therapeutic targets and ultimately improve clinical outcomes of patients with eosinophilia-mediated diseases. The Janssen lab serendipitously discovered that the mere perfusion of standard media without pharmacological additives over human eosinophils in vitro induced the release of intracellular calcium (Ca2+) reminiscent of chemokine-induced Ca2+ release well documented in the literature. The central focus of my doctoral research was to characterize this novel phenomenon of the perfusion-induced calcium response (PICR), and to determine its physiological role in the eosinophil extravasation process to inflamed tissue sites. In our first research objective, we optimized a protocol of eosinophil isolation directly from whole blood with emphases on maximizing population purity and yield efficiency while minimizing cell activation that could potentially interfere with secondary functional assays. For our latter two studies, we utilized real-time fluorescent confocal microscopy and immunofluorescence staining to investigate the PICR. We observed that the latency to the PICR post-perfusion was significantly shorter in eosinophils subjected to physiological rates of shear stress, suggesting a temporal-regulatory function of eosinophil mechanosensitivity. Furthermore, the disruption of the PICR via pharmacological inhibitors significantly reduced eosinophil motility by increasing the latency to cytoskeletal rearrangements (flattening onto substrate-coated surfaces, formation of membrane protrusions that explore the environment) necessary for cell migration out of the vasculature. Detailing the role of eosinophil sensitivity to the mechanical trigger of fluid shear stress expands upon the current paradigm of eosinophil recruitment and will contribute to the development of clinical strategies. / Dissertation / Candidate in Philosophy